31 research outputs found
Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2′-modified-4′-thionucleosides
AbstractGene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2′-modified-4′-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2′-fluoro-4′-thionucleoside and 2′-fluoronucleoside units as well as only 2′-fluoronucleoside units, while those prepared as combination of 2′-OMe nucleoside/2′-OMe-4′-thionucleoside and 2′-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity
Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5′-splice site mutation of human cathepsin A gene
Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5’-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5’-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene
Synergistic effect of combining theophylline and drugs that potentially elevate serum creatine kinase
An increase in the serum creatine kinase (CK) level is one of the side effects of theophylline;on rare occasions, the increase may be followed by rhabdomyolysis. Theophylline is often administered with drugs that potentially elevate the serum CK level (CK-elevating drugs) such as β-agonists and steroids. However, the effects of the combined treatment of theophylline and CK-elevating drugs have not been reported. We, therefore, retrospectively investigated the effects of combined treatment on the serum CK level, in391asthmatic outpatients.
In this study, the number and type of the CK-elevating drugs administered, and the serum levels of CK and theophylline, were investigated. The patients were divided into four groups:the theophylline-treated and CK-elevating drug-treated group, the theophylline-treated and non-CK-elevating drug-treated group, the non-theophylline-treated and CK-elevating drug-treated group, and the non-theophylline-treated and non-CK-elevating drug-treated group.
The theophylline-treated and CK-elevating drug-treated group showed about 100%higher serum CK levels (225IU/L) than any other group (102-124IU/L), and no increase in the serum theophylline level. This result indicates that there is a synergistic effect of theophylline and CK-elevating drugs on the serum CK level.
The combined treatment of theophylline and CK-elevating drugs induces a synergistic increase in the serum CK level, indicating not pharmacokinetic but pharmacodynamic interactions with these drugs
Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides
Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2'-modified-4'-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2'-fluoro-4'-thionucleoside and 2'-fluoronucleoside units as well as only 2'-fluoronucleoside units, while those prepared as combination of 2'-OMe nucleoside/2'-OMe-4'-thionucleoside and 2'-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity
Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein
Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms
Prevention of lethal hepatic injury in Long-Evans Cinnamon (LEC) rats by D-galactosamine hydrochloride
Repeated injections of D-galactosamine hydrochloride (GalN) increase the survival rate of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson’s disease. The aim of the present study was to investigate the mechanism of GalN for prevention of spontaneous lethal hepatic injury in LEC rats. MaleLEC rats were given a single subcutaneous injection of 300mg/kg of GalN or vehicle (0.9%NaCl) at 14weeks, and killed at 28 weeks of age. Next, 6-week-old male LEC rats were given weekly subcutaneous injections of 300 mg/kg of GalN or vehicle for 3 or 12 weeks, and their hepatic 8-hydroxydeoxy-2’-guanosine (8-OHdG), glutathione peroxidase (GPX), and catalase activities were measured. None of GalN-treated rats died of hepatic injury (0/12), whereas the mortality rate of control rats given 0.9% NaCl was 17% (2/12). GalN administration for 12 weeks decreased the hepatic 8-OHdG, and GalN administration for either 3 or 12weeks increased the glutathione peroxidase activity. GalN administration increased the serum level of alanine aminotransferase, and accelerated megalocytic degeneration of the hepatocytes. GalN treatment is effective in preventing lethal hepatitis in LEC rats and decrease of oxidative DNA damage by GalN plays an important role in increase of the survival rate